Project Summary Congenital heart disease (CHD) is the most common life threatening birth defect of the newborn period. CHD accounts for nearly one third of congenital defects world-wide, occurring in over one million newborns per year. Our understanding of the origins of CHD in people comes primarily from studies of Human Genetics and Epidemiology. Unfortunately, neither of these disciplines offers a complete picture. Indeed, we have come to recognize that the causes of CHD are multifactorial and depend on complex interactions between genetics and the environment. This proposal describes experiments aimed at better understanding these interactions through the use of an animal model. Sema3d mutant mice develop a type of CHD called total anomalous pulmonary venous return (TAPVR). The molecular mechanism of Sema3d in patterning the pulmonary veins has been well characterized in these mice. This mechanism suggests hypoxia and the molecular mediators of hypoxia may be potential environmental and genetic modifiers of the disease. Importantly, the Sema3d mutant mice develop TAPVR with incomplete penetrance, making them ideally suited for the study of factors that may increase or decrease the frequency of the disease, such as oxygen levels. The developing mammalian fetus receives limited oxygen from the mother through the placenta. In people, the delivery of oxygen to the placenta can be altered by altitude, anemia, or smoking. We will mimic the effect of these conditions by exposing pregnant mice to air with reduced oxygen content. We will test how this exposure influences the development of TAPVR in genetically sensitized mice. Furthermore, we will use CRISPR/Cas9 mutagenesis to target candidate genetic modifiers of Sema3d based on known interacting pathways. This approach allows a relatively high throughput means of testing molecular interactions with semaphorin signaling in the context of vascular patterning. Upon completion, these studies will provide important insights into environmental and genetic modifiers of pulmonary vein patterning resulting in TAPVR.